The purpose of this investigation is to initiate a biochemical study of genetic recombination in the eukaryotic organism Saccharomyces. For these studies DNA molecules will be constructed by recombinant DNA technologies which register through genetic and physical changes the occurence of genetic recombination events. The constructed recombination substrates will be plasmids which have two important features. First, the plasmid molecules will be capable of autonomous replication in either the yeast Saccharomyces or the bacteria Escherichia coli and will also encode selectable markers for purposes of plasmid identification in either organism. Thus, recombination deficient E. coli will be used to propagate plasmid molecules which will ultimately serve as substrates for recombination in yeast. The second important feature of the constructed plasmids will be that they contain an inverted repeated sequence in which the segments of the repeat will be different allelic forms of specific yeast genes. DNA molecules constructed with such inverted repetitions confer the novel possibility of distinguishing by a combination of physical and genetic assays the occurence of intramolecular gene conversion events with and without concomitant reciprocal recombination. Initially, the substrate molecules will be introduced by transformation into yeast cells in order to measure in vivo the rate of intramolecular recombination, the distribution of the different types of recombination events, and the sensitivity of the events to agents which affect genetic recombination. These studies serve not only to correlate the plasmid recombination events with the well studied, chromosomal recombination events, but also to provide insight into topological factors important to chromosomal recombination. The ultimate purpose of this proposal is to introduce constructed substrates into lysates of yeast cells in order to create an in vitro recombination system. An in vitro recombination system should provide a means to identify enzymes needed for genetic recombination.